The mammalian white matter is a major part of the brain where for many years potassium ions have been thought to be the only mediator of signaling between axons and glial cells, resulting mostly in passive glial depolarization. There are no known vesicular means of transmitter release in mammalian white matter. Yet recent studies have revealed that glial cells in white matter express glutamate receptors. This proposal examines how axons can dynamically activate glial receptors in this part of the brain. The white matter to be studied is the rat optic nerve. In Aim 1, we will examine whether glutamate is important in axon-glial signaling by using HPLC to detect activity-dependent release of glutamate from optic nerves, and by pharmacological dissection of glial glutamate receptor subtypes activated during nerve activity. We then focus on mechanisms of glutamate release and whether they are carrier-mediated. In Aim 2, we will provide evidence for carriers that might mediate glutamate release. We will use molecular biology to identify three major cloned transporters for glutamate (GLT, GLAST and EAAC1) in optic nerves. Then peptide-specific antibodies will be generated to examine the cellular localization of these transporters on glial cells and axons. In Aim 3, we will see if axonal and glial transporters mediate physiological glutamate release and whether their contributions can be sorted out. To distinguish glial and axonal release, we will use many techniques. These include selective manipulation of glial transporters in a nerve slice by patch-clamping, detection of axonal release by a detector patch, and high spatial resolution of glial and axonal release by immunogold analysis of D-aspartate at the electronmicroscopic level. The results will considerably extend our current understanding of neuron- glial interactions, and may lead to an understanding of potential new roles for glutamate transporters and glutamate receptors in regulating the development of mammalian white matter.